This application is directed to Challenge Area 14, Stem cells, and Specific Challenge Topic 14- NS-101, Reverse engineering human neurological disease: generation of stem cells from control and patient populations. The recent development of induced pluripotent stem cells offers an exciting opportunity to examine the cellular pathogenesis of a severe form of childhood epilepsy, SMEI, which is caused by mutations in a neuron-specific sodium channel gene, SCN1A. SMEI is an intractable disorder with onset between 6 months and 2 years of age. Our lab was involved in the initial identification of SCN1A mutations in 2001. Since then more than 500 mutations in this gene have been identified in sporadic and familial epilepsies. However, because of the neuron-specific expression pattern and the inaccessibility of neurons from patients, it has not been possible to obtain reliable information about the effects of these SCN1A mutations on the electrophysiological activity and firing patterns of affected cells. Identification of the specific neurons targeted for pathogenesis, and understanding of the cellular abnormalities that result from SCN1A mutations, are essential for the development of effect treatments. During the past few months, using the iPSC approach, we have been able to generate neurons containing a splice site mutation of SCN1A using fibroblast cultures prepared from a skin biopsy of an SMEI patient. We have been able to record action potentials from bipolar and pyramidal neurons. Using specific antibodies, we have identified GABAergic and other types of neurons in our culture. To continue this work, we have obtained access to a collection of control and patient fibroblasts. We propose to carry out a detailed characterization of firing patterns and electrophysiological properties of patient neurons and compare them with induced neurons from control individuals, to identify the unique properties of neurons expressing pathogenic SCN1A mutations. We will also investigate neuronal-specific splicing patterns of developmentally regulated alternative exons. We will study neurons carrying three classes of SCN1A mutations: splice site mutations, protein truncation mutations, and amino acid substitutions. These studies can be completed within 2 years and will provide the groundwork for developing a screening assay to identify pharmacological agents that can reverse the effects of SCN1A mutations in cultured cells and convert the mutant cellular phenotypes to those of normal controls. Cell lines and data generated during the course of this two year project will be shared with other investigators. The methods we develop will be applicable to many other neurological disorders. PUBLIC HEALTH RELEVANCE: Epilepsy is a common and debilitating neurological disorder, with a frequency of approximately 1 in 1,000 individuals. Many cases are resistant to currently available therapies. The severe genetic disorder SMEI has childhood onset and rapid progression and does not respond to treatment. Until now, it has not been possible to study the effects of SMEI mutations on nerve cells, because brain tissue from patients is not available for study. However, the newly developed technique of deriving neurons from skin biopsies means that SMEI and other neurological disorders can now be studied in molecular detail. We have succeeded in generating neurons from a patient with SMEI. These cells will be analyzed to determine the effects of the underlying mutation. After the molecular features of the abnormality are characterized, it may become possible to use these cultured neurons to screen for drugs which can prevent the progression of this disabling disease.